Withania somnifera is commonly known as Ashwagandha, Indian ginseng, Winter cherry and Ajagandha. It is mainly used in herbal formulations of the Ayurvedic or Indian system of medicine for treating memory loss, tumors, inflammation, arthritis, asthma and hypertension. The main constituents of ashwagandha are alkaloids and steroidal lactones. Among the various alkaloids, withanine is the main constituent. The other alkaloids are somniferine, somnine, somniferinine, withananine, pseudo-withanine, tropine, pseudo-tropine, 3-a-gloyloxytropane, choline, cuscohygrine, isopelletierine, anaferine and anahydrine. Two acyl steryl glucoside viz. sitoindoside VII and sitoindoside VIII have been isolated from root. The leaves contain steroidal lactones, which are commonly called withanolides. The withanolides have C28 steroidal nucleus with C9 side chain, having six membered lactone ring. Apart from leaves withanolides are also present in roots and berries.
Following patents/applications disclose processes for the preparation of Withania somnifera extract.
U.S. Pat. No. 6,713,092 discloses a process of making an Withania somnifera extract composition which comprises (a) providing root stock and leaves of a Withania somnifera plant which is about 1-2 years old, (b) extracting said root stock and leaves with an aqueous-alcoholic solvent in the presence of a exogenous saccharide, c) concentrating the extract under vacuum, (d) treating the residue with an apolar organic solvent to remove free withanolide A aglycones therefrom, (e) vacuum the insoluble residue of such treatment below about 60.degree. C. to provide a dry solid, and (f) pulverizing the solid under controlled temperature and humidity conditions, to obtain the desired powder product.
U.S. Pat. No. 6,153,198 discloses a process of making the extract of Withania somnifera which comprises (a) providing root stock of a Withania somnifera plant which is about 1-2 years old, (b) extracting said root stock with an aqueous-alcoholic solvent, (c) concentrating the extract under vacuum, (d) treating the residue with an apolar organic solvent to remove free withanolide A aglycones therefrom, (e) vacuum drying the insoluble residue of such treatment below about 60° C. to provide a dry solid, and (f) pulverizing the solid under controlled temperature and humidity conditions, to obtain the powder product. The aqueous-alcoholic solvent used is water-methanol or water-ethanol and the organic solvent used is chloroform or ethyl acetate.
US 20040033273 discloses a method for obtaining a composition having immune stimulating activity or anti-tumor activity from Withania somnifera comprising: (a) contacting Withania somnifera plant or plant part with a first medium polar solvent to produce a particulate suspension; (b) clarifying the particulate suspension to produce a clarified first solution and a first residue; (c) evaporating the solvent from the first clarified solution to produce a fraction, denoted fraction A; (d) resuspending the first residue in a second polar solvent thereby producing a second solution and a second residue; (e) clarifying the second solution to produce a second clarified solution; (f) evaporating the second polar solvent from the second clarified solution to produce a fraction, denoted fraction B; (g) resuspending the second residue in a third solvent more polar than the second polar solvent thereby producing a third solution and a third residue; (h) clarifying the third solution to produce a third clarified solution; (i) evaporating the third solvent from the third clarified solution to produce a fraction, denoted fraction C; (j) combining fractions A, B and C to produce an extract; (k) resuspending the extract in a solution to produce a fourth alkaline solution; and (l) fractionating the fourth solution with a non polar solvent and removing the solvent to produce a composition having immune stimulating activity or anti-tumor activity. The first residue is resuspended in a solvent having about 50% ethanol or about 40 to 60% isopropyl alcohol. The first medium polar solvent comprises acetone, tetrahydrofuran or ethylacetate. The second solvent comprises a mixture of water and isopropyl alcohol (IPA).
U.S. Pat. No. 7,108,870 discloses a process for isolation of withaferin-A from plant materials, said process comprising the steps of: (i) extracting the plant materials in an aqueous alcohol extraction solvent, (ii) defatting the extract, as obtained in step (i), with partitioning with n-hexane followed by chromatographic separation to obtain a withanolide preparation, (iii) portioning out withanolide aglycones from the withanolide preparation, as obtained in step (ii), into chloroform followed by evaporation of said chloroform to obtain a chloroform extract, and (iv) dissolving the chloroform extract as obtained in step (iii) in methanol followed by chromatographic separation to obtain withaferin-A.
The extraction is performed using a 60:40 methanol:water extraction solvent.
The prior art discusses crude Withania somnifera extract preparation methods which are not sufficient to produce a biologically active product. Accordingly, it is desirable to develop a process for the preparation of Withania somnifera fraction which is rich in withanolides.
Vaccines based on polysaccharide antigens are well known. The capsular polysaccharides of Neisseria meningitidis are attractive vaccine candidates because they constitute the most highly conserved and most exposed bacterial-surface antigens. The use of capsular polysaccharides as immunoprophylactic agents against human disease caused by encapsulated bacteria is now firmly established. The capsular polysaccharides of the meningococcus are negatively charged and are obtained in a high molecular weight immunogenic form by precipitation. Meningococcal polysaccharide vaccines are efficacious to protect from meningitis disease in adults, but cannot provide full protection to infants under the age of 5.
The duration of protection elicited by the meningococcal polysaccharide vaccines is not long lasting in adults and children above four years of age. For children from one to four years old the duration of protection is less than three years. Protective immunity to encapsulated bacterial pathogens such as N. meningitidis is principally mediated by the reaction between antibody and capsular polysaccharide epitopes. In encapsulated gram negative bacteria, protection results primarily from a direct complement-mediated bactericidal effect.
Vaccines have been prepared from the capsular polysaccharides of Neisseria meningitidis (groups A, C, W-135 and Y). These and other polysaccharides have been classified as T cell independent type 2 (TI-2) antigens based on their inability to stimulate an immune response in animals that carry an X-linked immune B-cell defect. TI-2 antigens tend to be characterized by high molecular weight, multiple repeat epitopes, slow degradation in vivo and a failure to stimulate major histocompatibility complex (MHC) type II mediated T-cell help. TI-2 antigens generally are incapable of stimulating an immune response in neonatal humans under 18 months of age.
Polysaccharide antigens are T-lymphocyte independent antigens and therefore, IgG response elicited by the antigens is very limited. Moreover, the immune response elicited by polysaccharide antigens is characterized by low immunological memory and therefore the immunity induced by the use of such antigens is short lived. Further, young children respond poorly to polysaccharide antigens and therefore vaccines containing them have not been proven to be effective in young children. To enhance the immune response elicited by polysaccharide antigens, aluminium salts have been known to be used as adjuvants in vaccines containing these antigens. However, aluminium containing vaccines, when injected, have been reported to cause undesirable local reactions.
U.S. Pat. No. 6,645,495 discloses a composition comprising an antigen or a nucleic acid encoding an antigenic peptide or protein, a saponin adjuvant such as Quil-A, QS-7 and QS-21, and an excipient selected from a group consisting of beta cyclodextrin and human serum albumin.
However, Quillaja saponins have been found to be unstable in aqueous phase (Sun, Hong Xiang et al., 2009, Vaccine, vol. 27, p. 1787-1796). Quillaja saponins have strong haemolytic effect and also cause undesirable local reactions like swelling, skin degeneration. Even the deaths of mice used in the experiments have been reported due to presence of Quillaja saponins (Ronnberget et at., vaccine, vol. 13, 1995, 1375-1382.)
The Indian patent application No. 1253/MUM/2003 published on 20 Jan. 2006 discloses the use of Withania somnifera extract as an adjuvant in vaccines. However, it does not provide adjuvant composition for vaccines based on polysaccharide antigens such as meningococcal vaccine prepared from polysaccharides of Neisseria meningitides. Further, the disclosed crude extract is known to contain a large proportion of polysaccharides that could adversely affect the bioavailability of the active compounds present therein. Moreover, to attain the desired adjuvanticity, large quantity of the extract is required to be used in the vaccine.
Accordingly, it is desirable to develop highly pure adjuvant which has greater immune adjuvant activity than crude Withania somnifera extract. Further, there is also need of a vaccine comprising this adjuvant and vaccine antigen which provides enhanced immunological response in the host.